The broad aim of the proposed study is to understand the mechanism of disease caused by intracellular bacteria. The tribe Ehrlichieae are a group of pathogenic rickettsiae and obligate intracellular parasites of macrophages or granulocytes. They multiply in the membrane-lined vacuoles that do not fuse with lysosomes. As ehrlichial organisms multiply, their vacuolar membrane must be remodeled to accommodate spacial and metabolic needs of the parasite, while constantly maintaining the inhibition of lysosomal fusion. The nature, mechanism, and consequence of the membrane remodeling are unknown. Hypothesis 1 is that the inclusion membranes are selectively modified by insertion of specific ehrlichial proteins including antigens, consequently, the modified membrane fails to fuse with lysosomes. Inhibition of ehrlichial antigen translocation thus abrogates the inhibition of lysosomal fusion. Our hypothesis 2 is that the ehrlichial antigens are shuttled to the surface of the infected macrophage which is lysed by antibody-dependent cell- mediated cytotoxic (ADCC) mechanism or with the antibody and complement. Thus, parasite antigens inserted into the host membrane have a key role in pathogenesis and immune response in the ehrlichial disease. We found strong evidence that supports a part of these hypotheses, i.e., demonstration of ehrlichial antigen on the surface of infected cells, ADCC of the infected cells, and likely abrogation of inhibition of lysosomal fusion with anti-Ehrlichia-antibody. Specific Aims of this research are: Aim 1: Test hypothesis 1 by identifying molecular sizes and numbers of ehrlichial proteins (antigens) translocated into the host plasma membranes. Aim 2: Test hypothesis 1 by identifying the mechanism and intracellular site of ehrlichial antigen insertion by immunocytochemistry and EM-autoradiography. Aim 3: Test hypothesis 1 by evaluating the effects of anti-ehrlichial monoclonal antibodies on vacuolar acidification and lysosomal fusion, examining lysosomal fusion with vacuoles containing ehrlichial antigens expressed on the host cell surface, and examining translocation of ehrlichial proteins or antigens in infected macrophages treated with interferon-gamma, concanavalin A, calcium ionophore A23187, and oxytetracycline. Aim 4: Test hypothesis 2 by examining ADCC using 51Cr-labeled infected cells and measuring complement and antibody-dependent lysis of infected cells. The project not only contributes in developing rational treatments and prevention strategies for ehrlichial diseases but also may reveal general principles of the pathogenesis of the disease caused by intracellular parasites.